In-vitro evaluation of the anti-cariogenic effect of a hybrid coating associated with encapsulated sodium fluoride and stannous chloride in nanoclays on enamel

Abstract Objective The aim of this study is to test, in vitro, the anti-cariogenic effect of experimental hybrid coatings, with nano clays of halloysite or bentonite, loaded with sodium fluoride or with a combination of sodium fluoride and stannous chloride, respectively. Methodology The varnish Fluor Protector (1,000 ppm of F-) was used as positive control and no treatment was the negative control. Enamel specimens (5 mm × 5 mm) were obtained from bovine teeth. The specimens (n=10) had their surfaces divided into two halves (5 mm × 2.5 mm each), in which one half received one of the treatments (Hybrid; Hybrid + NaF; Hybrid + NaF + SnCl2; Hybrid + NaF Loaded; Hybrid + NaF + SnCl2 Loaded). The specimens were submitted to a cariogenic challenge using a biofilm model (S. mutans UA159, for 5 days). Enamel surfaces both under and adjacent to the treated area were analyzed for mineral loss and lesion depth, by transverse microradiography. The pH of the medium was measured twice a day, and the fluoride release was analyzed. Additional specimens were submitted to confocal analysis. Results Data were statistically analyzed by two-way ANOVA followed by Tukey test (α=0.05). None of hybrid groups were able to reduce the lesion depth; the Hybrid + NaF group, however, was able to reduce mineral loss differing from the negative control (p=0.008). The groups showed no significant difference in the pH measurement and fluoride release. Confocal analysis confirmed that for all groups the biofilm growth was similar. Conclusion None of the hybrid groups reduced lesion depth, but the Hybrid + NaF group was able to promote protection against mineral loss.


Introduction
Fluoride therapy is one of the main strategies used for caries control. There is a wide range of fluoridated products available, for either at-home or professional use. The mode of action of fluoride against caries is post eruptive and local. 1 When constantly present in dental biofilm, it can reduce demineralization and enhance remineralization. 2 Monovalent fluoride compounds, such as sodium fluoride are the most common. Stannous fluoride, by contrast, is a polyvalent fluoride compound containing metal cation, which has also shown to protect against caries. 3,4 Technological advances have significantly improved some of the drawbacks associated with the use of stannous fluoride, such as its low stability and tooth staining. 3 The anti-cariogenic mechanism of Sn 2+ relies on two properties inherent to this cation, including antimicrobial function and high affinity with the apatite surfaces. The antimicrobial action takes place through the inhibition of microbial enzymes involved in the transport and metabolism of glucose. 4 This appears to be a stable activity due to stannous good oral substantivity, especially in the biofilm. 4,5 Stannous can also interact with the tooth surfaces, forming a protective layer composed of a variety of compounds, 6 which can also interfere with the biofilm architecture. 4 The use of professionally applied fluoride products is one preventive strategy used around the world in private and public health settings. The biannual application of fluoride varnish has shown to promote a decrease in caries incidence over two years, especially on high-risk patients. 7,8 The varnishes have the ability to adhere to tooth surfaces, prolonging the contact time between fluoride and enamel. After application of the varnish, saliva bathes the varnish and dissolves the fluoride salt, allowing fluoride ions to diffuse out of the varnish and become absorbed into fluoride reservoirs within oral soft tissues, plaque, and teeth. 9 Although the mode of action of the fluoride varnish against caries is not fully understood, it is known that the bioavailability of fluoride is fundamental for controlling the progression of this condition. 9,10 Another possible approach to protect the tooth against cariogenic challenges would be the use of a smart hybrid coating material, which contains a stock of active agents encapsulated in nano clays that are available under demand for preventing caries.
A previous study showed that experimental hybrid coatings can chemically adhere to the dental surfaces after an alkaline treatment, causing dentin tubule occlusion. 11 These coatings are known to have antistaining properties, in addition to thermal, chemical, and biological resistance, which are characteristics attributed to their inorganic components. They also have flexibility, film-forming ability, and possibility of adhesion, which are features related to their organic components. 11 The use of this hybrid coating appears to be promising for many dental applications and has not been fully explored. To our knowledge, there are no published studies testing the hybrid coating as a possible preventive approach against caries.
Despite the benefits, hybrid coatings present some shortcomings, such as the development of pores and defects when constantly challenged, mechanically and chemically. In view of this, the idea of including agents with a potential protective effect against caries in these coatings is worth considering.
Special attention should be directed toward fluoride and the combination of fluoride and stannous, since they could potentially act on any defects, reducing demineralization and enhancing remineralization. 3,12 For these agents to act on demand, they could be loaded into nano clays, from which Fand Sn 2+ would be slowly released into the oral environment at the time of the cariogenic challenge. This release would not only promote minerals deposition on enamel surface, but also potentially act on enamel subsurface, similarly to the fluoride varnish. 13 A previous study 14 showed the feasibility of loading sodium fluoride and stannous chloride into nano clays of halloysite and bentonite, with posterior release of these ions on different mediums (acidic and neutral).
The aim of this study is to test, in vitro, the anticariogenic effect of experimental hybrid coatings with sodium fluoride or with the combination of sodium fluoride and stannous chloride, which were loaded into nano clays of halloysite and bentonite, respectively. A dynamic biofilm model was used. The null hypotheses were: 1) the experimental hybrid coatings would not differ from the negative control regarding enamel mineral loss and lesion depth assessed after the cariogenic challenge on the treated surface; 2) enamel mineral loss and lesion depth would not differ between the experimental hybrid coatings and the negative control on the surface adjacent to treatment.
In-vitro evaluation of the anti-cariogenic effect of a hybrid coating associated with encapsulated sodium fluoride and stannous chloride in nanoclays on enamel J Appl Oral Sci. 2022;30:e20210643 3/12

Methodology Study Design
The study protocol was approved by the local ethics committee in research (#1406440799R001).
This study followed a complete randomized design, with two experimental factors: treatments and specimen area (n=10). 1) Treatments, at 7 levels: The treatments were tested using a dynamic cariogenic model with bovine enamel specimens (n=10). The enamel specimens (5 mm × 5 mm) were prepared and had their surface divided into two halves (5 mm × 2.5 mm each), one in which the treatment was applied (under-treatment), and the other that was adjacent to the treated area (adjacent to treatment).
The specimens were submitted to the cariogenic challenge, using a biofilm model. The response variable was the mineral content change, measured by transverse microradiography and expressed by the total mineral loss and average lesion depth.
Sample size calculation A pilot study (not shown) was performed to determine the number of specimens per group. The sample size calculation was made on SigmaPlot 12.0.
ANOVA Sample size was used, considering an effect size of -3784 for mineral loss and -119 for lesion depth, α=0.05 and a power of 0.80, obtaining a sample size of 5. Considering this and the previous studies that employed a similar methodology, we adopted n=10.

Specimen Preparation
A total of 70 enamel slabs (5 mm × 5 mm) were sectioned from the crowns of the bovine incisors and was analyzed with a stereoscopic microscope to certify that they were free from demineralization, cracks, or any other defects. Bovine incisors were used instead human due to their chemical and physical similarities. 15,16 After collection and during the preparation process, the teeth were stored in 0.1% thymol solution under refrigeration at 4ºC.
The bottom and top (enamel) sides of the slabs were sequentially ground flat using silicon carbide grinding papers (#600, #1200, #2400 for 15 s, 25 s, and 30 s, respectively) (RotoPol 31 / RotoForce 4, Struers, Cleveland, Ohio, USA). The top side was serially polished up to a 4,000-grit grinding paper, followed by 1-μm diamond polishing suspension. Afterwards, they were sterilized in a steam autoclave, at 121ºC for 30 min followed by 10 min air-drying at sub-atmospheric pressure.
All the treated specimens were fixed in the lid of 24well culture plates and were submitted to a cariogenic challenge, using a biofilm model 17 for 5 days.

Hybrid coating
T h e h y b r i d c o a t i n g w a s p r e p a r e d a s previously described, 11,18 contained a gammaa m i n o p r o p y l t r i e t h o x y s i l a n e ( γ -A P S ) ,

Experimental group Abbreviation Components
Negative control with the aid of a disposable applicator and with a 60 s wait for complete cure. For the hybrid coating groups, the protocol used was established in previous studies 11,14 as follows: firstly, an alkaline surface treatment was performed with 0.05 M NaOH solution (pH of approximately 12.9) for 10 min, followed by rinsing with deionized water and drying. Then, the experimental hybrid solutions were applied with disposable applicator, in two layers. After each layer, the solution was allowed to dry for 4 min followed by application of heat source with a light curing device (Valo, Ultradent, South Jordan, UT, USA) for 60 s, at irradiance of 1,000 mW/cm 2 , with 1 mm apart, approximately, to complete the cure of each hybrid layer.

Biofilm model
The cariogenic biofilm model used was based on a previous study, 17 with modifications according to preliminary test results (data not published).

Streptococcus mutans UA159 reference strain 19
was used in the experiment. Depending on the experimental phase, the content of the media was 1% glucose, 1% sucrose or 0.1% glucose, as described below. S. mutans colonies were transferred to Brain Heart Infusion (BHI) supplemented with 1% glucose and were incubated for 18 h -24 h at 37°C, 5% CO 2 to reactivate the microorganisms. Slabs were first individually positioned in cell plates cover ( Figure   3), then they were coated with human saliva, and a salivary pellicle was formed by being incubated in filter-sterilized clarified human whole saliva (IRB #1406440799R001) for 30 min, at 37ºC. After salivary pellicle formation, they were placed in 1.8 mL of the

Confocal Scanning Laser Microscopy (CSLM) Analysis
Additional specimens were used for the confocal analysis to determine the biofilm morphology, using

Fluoride Analysis
Aliquots of 1 ml of all media were collected twice a day, immediately after the culture media was changed.
The aliquot was then mixed 1:1 with TISAB II (0.5 mL of sample + 0.5 mL of TISAB II) and analyzed for fluoride by comparison to a similarly prepared standard curve (1 mL standard + 1 mL TISAB II) using an ion-selective electrode (Orion Research, Boston, MA, USA). Fluoride data were calculated as µg F/mg (mean amount per specimen).

Statistical Analysis Data of Mineral Loss and Lesion Depth were
statistically analyzed and were individually tested for normal distribution and homoscedasticity with Shapiro-Wilk and Brown Forsythe test, respectively. Considering that the data followed a normal distribution, a two-way repeated measurements ANOVA, followed by Tukey post-hoc test, was used for assessment. A 5% of level of significance was considered and the Sigma Plot (12.0) software was used for calculations.

Results
The microradiography ( Figure 5) results are shown in For mineral loss, on the under-treatment surface, the Fluor Protector was the group that showed the lowest mineral loss, significantly differing from the negative control (p<0.001) and the other groups (p<0.001).
The Hybrid + NaF group showed significant difference from negative control (p=0.008), but not from the groups with the loaded agents (p>0.005). The Hybrid + NaF + SnCl 2 group, Hybrid group, and the groups

Discussion
The first null hypothesis of the study, which stated that the experimental hybrid coatings would not differ from the negative control as regarding mineral loss and lesion depth of the enamel assessed after the cariogenic challenge on the treatment surface, was rejected since the experimental hybrid coating with NaF was able to reduce the mineral loss of the enamel in more than 50% when compared to the negative control group. This could be attributed to the fluoride present in the hybrid coating, which was slowly released and was able to protect the surface against cariogenic acid. The Hybrid coating group, however, was not capable of reducing lesion depth significantly.
The application of the hybrid coating solution on a polished enamel surface could have interfered with the mechanical retention of the coating during cycling, leading to its partial loss. Thus, the remaining material was not able to physically protect the surface against the challenges. Whereas, when the coating was loaded with fluoride, it served as a fluoride deposit, releasing this ion onto the lesion, optimizing remineralization.
This low retention of the hybrid coating to the enamel surface was also observed in a previous investigation, 14 in which an initial demineralization was performed in the specimens to increase the retention of the coating. This initial demineralization simulates an already established lesion, which received the treatment with the coating to slow down its progression.
To increase the hybrid adhesion, the authors suggests that a micro-retention with phosphoric acid, or with citric acid in high concentration may be created in future studies. On a previous study 14 from our group -using a different model, with more aggressive acid and with more exposure to the challenge than the one used in this study -the hybrid coating was able to significantly protect the enamel and dentin surface against dental erosion. In that study the specimens had been previously eroded when the hybrid was applied, and this micromechanical retention could have increased the adhesion of the coating.
A different behavior of the hybrid coating was expected in these different models, as it is known that dental caries and dental erosion are distinct processes.
Dental erosion results in a demineralized surface, with the acid also affecting a near-surface layer; considered mainly a surface phenomenon. By contrast, dental caries in its initial process is a subsurface phenomenon, in which the destructive effects occur on the surface, but mostly within the subsurface region. 21 The fluoride varnish (Fluor Protector) was the only group able to protect the under-treatment area The second null hypothesis of this study was accepted since none of the groups were able to significantly protect the area adjacent to treatment.
Although the fluoride varnish and the Hybrid + NaF loaded showed significantly lower lesion depth in relation to the other groups, they did not differ from negative control. When the areas were compared, the varnish and Hybrid + NaF groups were able to promote a smaller lesion depth on the under-treatment surface.
Furthermore, these groups and the loaded groups (Hybrid + NaF Loaded and Hybrid + NaF + SnCl 2 Loaded), reduced the mineral loss of the same area when compared with area adjacent to treatment. We believe that this slightly better protection was due to the action of the ions that were released by the clays presented on the remnants of coating. Protector promoted better enamel protection against demineralization, the results of our study showed that Hybrid + NaF coating was the best treatment when compared to the other hybrid treatments. It has the potential to be used in future studies as a protective product against demineralization, given that its retention to the enamel surface is improved.
Surprisingly, the groups with nano containers (halloysite and bentonite) loaded with fluoride and stannous ions did not show significant effect under the circumstances of this study, when compared to the control group. It is already known that the addition of nano containers to polymeric films may improve their mechanical resistance, without compromising the performance of the material. 34 The addition of halloysite clay nanotubes loaded with chlorhexidine into bonding agents did not change their viscosities. 35 In this study, however, an increased viscosity of the hybrid coating was visually observed, which lead us to hypothesize that the amount of nano clays added was excessive, to a point that its adhesion to the enamel substrate was further jeopardized. The more viscous the hybrid solution, the harder it would be for it to penetrate irregularities of the enamel, allowing the coating to be more easily detached from the enamel surface. Thus, the ions had short time to interact with the enamel surfaces, showing lower protective effect.
Additionally, the hybrid coating was a dense network and may have acted as a barrier, not properly allowing the releasing of ions, even for nonencapsulated agents. It is known that, after the cure of the hybrid solution, a crosslinking occurs resulting in chemically stable bonds with the tooth surface; allowing the hybrid coating to act as a mechanical barrier against the acid challenge. 14 As described on previous study, 14 the material needs an adequate cure to achieve good properties, which should be performed at high temperatures. 36 We believe that the cure performed in this study may not have been enough to promote satisfactory adhesion of the material to the surface. Therefore, more studies are necessary to improve the cure of the hybrid coating.
Moreover, further studies need to be conducted to enhance the adhesion of this material to dental substrates, considering that this treatment would be indicated for professional use, especially in areas of the teeth that already had some demineralization and irregularities. A less aggressive model should be used in future studies to test the hybrid coatings, which might give more opportunities to the ions to be released slowly and be able to protect the enamel surface.

Conclusion
Fluor Protector showed greater protection against cariogenic challenge. None of the hybrid coatings treatments reduced lesion depth; nevertheless, the Hybrid with NaF prevented enamel mineral loss. Future studies aiming to improve the retention of the hybrid coatings on sound enamel are needed, in order to optimize its effect.